Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/48—Reproductive organs
  • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/38—Albumins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

• the present technology generally relates to placental compositions, methods of medical treatment using placental compositions, and methods of making placental compositions.
• the present technology relates to methods and products that facilitate or improve wound healing including, for example, compositions, cryopreserved compositions, methods for promoting one or more of angiogenesis, reduction of inflammation, reduction of scar formation, reducing protease activity, promoting cell migration, promoting tissue regeneration, and inhibition of free radical oxidation.
• EGF epidermal growth factor
• PDGF platelet derived growth factor
• bFGF basic fibroblast growth factor
• VEGF vascular endothelial growth factor
• placental membranes for burns and other types of wounds originated more than 100 years ago (as reviewed by Kesting et al., 2008).
• Placental membranes contain components that are present in skin and are required for wound healing such as extracellular matrix, growth factors, and cells, including MSCs (mesenchymal stem cells), for example, that are responsible for orchestrating the process of wound healing.
• MSCs mesenchymal stem cells
• placental membranes such as amniotic membranes for burns was recorded in a number of published reports; however, the use of placental membranes for large surface area burns is limited due to challenges in providing sufficient placental membranes to cover large areas.
• Tunneling wounds are characterized by "tunnels" that channel from the wound into or through the muscle or subcutaneous tissue and can have one or more tunnels with varying length or depth.
• causes for tunnel wound development include infection, prolonged inflammation for chronic wounds, pressure and shear forces that are concentrated where tissue layers meet, inadequate drainage absorption due to insufficient wound packing and degradation of newly granulated tissue due to too much wound packing.
• Dressings in a sheet form are not suitable for tunnel wounds, since the dressing will not cover the wound surface, and if placed inside the tunnel, will exert too much pressure and degrade newly granulated tissue.
• the present technology provides one or more placental compositions comprising placental cells and other placental components derived from placental tissue, e.g., a whole placenta or portion thereof.
• the other placental components may comprise one or more therapeutic factors, extracellular matrix components, and the like.
• the composition(s) can be obtained by mechanical manipulation (e.g., dissection or mincing or homogenization), enzymatic digestion or combinations thereof.
• a placental tissue can optionally be an amnion, chorion, a mixture of amnion and chorion, or other tissue described herein, including, for example, umbilical cord tissue and Wharton's jelly.
• the present technology also provides a method of making one or more placental compositions.
• the present technology also provides one or more methods of treating a whole or partial tissue injury or defect (e.g., wound or burn) comprising administering to a patient (human or animal) in need thereof one or more placental compositions of the present technology.
• the present technology in some aspects also provides one or more methods of regenerating tissue comprising administering one or more placental compositions of the present technology to a patient (human or animal) in need thereof.
• the placental compositions of the present technology comprises one or more therapeutic factors set forth, for example, in Table 1 , Table 2, or Table 5, among others.
• the placental cells utilized in the practice of the present technology comprise stromal cells such as MSCs (mesenchymal stem cells).
• compositions of the present technology are produced by a parallel processing method that comprises:
• a first placental tissue e.g., amniotic or chorionic tissue
• a second placental e.g., amniotic or chorionic tissue
• the first placental tissue and the second placental tissue are autologous to each other, for example, derived from the same donor.
• compositions of the present technology are produced by a serial processing method wherein the second placental tissue is derived from the first placental tissue after obtaining the placental cells from the first placental tissue.
• a first chorionic tissue may be retained after isolating a population of cells thereof, and then disrupted to form a dispersion. The dispersion may then be combined with the placental cells.
• the step of isolating the placental cells can comprise the step of contacting the first placental tissue (e.g., amnion or a chorion or a chorion lacking trophoblasts) with a digestive enzyme, such as a collagenase type II, among others.
• a digestive enzyme such as a collagenase type II
• the first placental tissue is exposed to a limited digestion with an enzyme such as collagenase type II (e.g., exposure for less than about 1 hour; alternatively about 20 minutes; or alternatively about 10 minutes).
• the placental tissue (from which the placental composition of the present technology is produced) is chorionic tissue depleted of trophoblasts by treatment with a digestive enzyme such as dispase followed by physical removal of the trophoblasts.
• a digestive enzyme such as dispase followed by physical removal of the trophoblasts.
• the method of making one or more placental compositions of the present technology comprises the steps of:
• obtaining a placental (e.g., amniotic or chorionic) tissue e.g., amniotic or chorionic tissue
• the method of making one or more placental compositions of the present technology comprises the steps of:
• the disrupting step comprises homogenizing the collagenase exposed placental tissue. In another aspect, the disrupting step comprises mincing the collagenase exposed placental tissue. Other forms of disruption are also envisaged in the practice and performance of the present technology.
• the one or more methods of the present technology further comprises a step of cryopreserving the placental composition wherein, after thawing, at least 40% of the placental cells in the placental composition are viable.
• cryopreserved placental tissue compositions comprising at least one disrupted placental tissue having:
• tissue pieces comprising (i), (ii), (iii) or combinations thereof; wherein after subsequent thawing of the cryopreserved placental tissue composition, greater than about 40% of the placental cells are viable and the composition is depleted in functional immunogenic cells (for example, as compared to the placental tissue composition prior to such cryopreservation and subsequent thawing).
• cryopreserved placental tissue compositions comprising:
• At least one disrupted placental tissue including:
• tissue pieces comprising (i), (ii), (iii) or combinations thereof;
• cryopreservation agents wherein after subsequent thawing of the cryopreserved placental tissue composition, greater than about 40% of the placental cells are viable and the composition is depleted in functional immunogenic cells; and wherein the one or more placental cells, therapeutic factors, extracellular matrix components, or combinations thereof are present in an amount effective to provide at least one therapeutic benefit.
• the present technology provides at least one process for preparing one or more placental tissue compositions comprising the steps of: (a) providing a placental tissue comprising placental cells, therapeutic factors, and extracellular matrix components; (b) disrupting at least a portion of the placental tissue to form a placental dispersion comprising placental tissue pieces, placental cells, therapeutic factors and extracellular matrix components; and (c) cryopreserving the placental dispersion to form a cryopreserved placental tissue composition, wherein after subsequent thawing of the cryopreserved placental tissue composition, greater than about 40% of the placental cells are viable and wherein the composition is depleted in functional immunogenic cells.
• the present technology provides at least one process for preparing a placental tissue composition comprising the steps of:
• cryopreserved placental tissue composition wherein after subsequent thawing of the cryopreserved placental tissue composition, greater than about 40% of the placental cells are viable and wherein the composition is depleted in functional immunogenic cells.
• the present technology provides a composition made by the one or more of the processes disclosed herein.
• cryopreserved placental tissue composition comprises:
• a. disrupted placental tissue having:
• tissue pieces comprising (i), (ii), (iii) or combinations thereof; and b.one or more cryopreservation agents,
• placental tissue composition wherein after cryopreservation and subsequent thawing of the placental tissue composition, greater than about 40% of the placental cells are viable and the composition is depleted in functional immunogenic cells; and wherein the one or more placental cells, therapeutic factors, extracellular matrix components or combinations thereof are provided in an amount effective to do one or more of the following:
• cryopreserved placental tissue compositions comprising:
• a minced placental tissue dispersion including:
• tissue pieces comprising (i), (ii), (iii) or combinations thereof;
• cryopreserved placental tissue composition wherein after subsequent thawing of the cryopreserved placental tissue composition, greater than about 40% of the placental cells are viable and wherein the composition is substantially depleted in functional immunogenic cells.
• the composition may be stored for an extended period of time prior to subsequent thawing.
• the extended period of time can be from about 6 months to about 36 months or more, alternatively from about 6 months to at least about 24 months or greater, alternatively from about 6 months to at least about 12 months or greater, alternatively from about 6 months to about 10 months, alternatively from about 6 months, alternatively from about 3 months to about 6 months, alternatively from about 1 month to about 3 months, including other monthly and day derivations thereof for the various time periods described herein.
• the viability of the tissue cells is substantially maintained upon subsequent thawing. In further embodiments, the viability of the tissue cells is substantially maintained for at least 24 months when stored frozen.
• the present technology provides at least one method of treating a tunnel wound of a subject (human or animal) in need thereof comprising administering to the site of the tunnel wound one or more placental compositions as described herein.
• the present technology provides at least one method of treating a wound or tissue defect of a subject (human or animal) in need thereof comprising the step of administering at least one placental composition as described herein.
• the amount of the composition is effective to reduce inflammation upon administration.
• the amount of the composition is effective to increase angiogenesis upon administration.
• the amount of the composition is effective to provide anti-oxidant conditions upon administration.
• the present technology provides methods of promoting angiogenesis in a whole or partial wound or tissue defect comprising the steps of administering a placental composition as described herein in an amount effective to promote angiogenesis.
• the present technology provides methods of preventing or reducing formation of scars comprising administering to the site in need thereof one or more placental compositions as described herein in an amount effective to prevent or reduce formation of scars.
• the present technology provides methods of improving wound healing comprising administering one or more placental compositions as described herein to a subject (human or animal) in need thereof, and wherein the placental composition is provided in an effective amount to promote increased expression of one or more therapeutic factors.
• the present technology provides methods of directly or indirectly stimulating tissue regeneration comprising administering one or more placental compositions as described herein to a subject (human or animal) in need thereof, and wherein the placental composition is provided in an effective amount to promote increased expression of one or more therapeutic factors.
• the present technology provides a method of reducing protease activity at a site in need thereof, comprising administering to the site an amount of one or more placental compositions as described herein effective to reduce the protease activity upon administration to the site.
• the present technology provides at least one composition comprising: a) a thawed cryopreserved placental composition; and b) a carrier.
• the present technology provides at least one kit for treating a whole or partial wound or tissue defect comprising: a) at least one cryopreserved placental tissue composition in or on or associated with at least one pharmaceutically acceptable container; and b) instructions for administering the placental tissue composition for treating the whole or partial wound or tissue defect.
• Figure 1 depicts recovery of viable cells/gram of chorionic tissue isolated by digestion.
• Figure 2 depicts quantity of viable and non-viable cells isolated by digestion.
• Figure 3 depicts cell viability, before and after a freeze-thaw cycle of a placental product.
• Figure 4 depicts the level of viable cells in a placental product made with or without a digestion step of the present technology before homogenization.
• Figure 5 depicts cell phenotype of cells in a placental composition of the present technology by staining with cell surface markers CD166 (5B), CD105 (5C), and CD45 (5D) as compared with isotype control (5A).
• Figure 6 depicts cell viability using various cryoprotectants.
• Figure 7 depicts cell viability of placental composition of the present technology after storage in different cryopreservation solutions and at different time points after freezing.
• Figure 8 depicts placental tissue weight and quantity of live cells recovered following collagenase treatment of various incubation times.
• Figure 9 depicts the recovery of viable cells isolated by digestion and homogenization of amniotic membrane.
• Figure 10 depicts expression of bFGF (10A) and VEGF (10B) in placental compositions of the present technology for 14 days in culture.
• Figure 1 1 depicts increase in VEGF production when a placental composition of the present technology is exposed to hypoxic conditions.
• Figure 12 depicts VEGF (12A) and bFGF (12B) content in placental compositions of the present technology before freezing and after freeze/thaw.
• Figure 13 depicts expression of IFN-2a (13A) and TGF-B3 (13B) in placental compositions of the present technology.
• FIG. 14 depicts TGF-B3 in multiple lots of a placental composition of the present technology.
• Figure 15A depicts detection of BMP-2, BMP-4, BMP-7, PLAB, and PLGF and Figure 15B depicts detection of IGF-1 in placental compositions of the present technology as derived from the chorionic membrane.
• Figure 16 depicts passage 2 cells isolated and expanded from bone marrow (16 A) or a placental composition derived from the chorionic membrane (16B) compared to cells isolated and expanded from a placental composition derived from chorionic membrane after osteoinduction (16C).
• Figure 17 depicts the quantity of viable cells (1 A) and the % cell viability (17B) in multiple lots of minced placental composition of the present technology.
• Figure 18 depicts the response to an inflammatory environment by TNF-a inhibition of minced and digested placental compositions of the present technology.
• Figure 19 depicts the inhibition of elastase by minced placental compositions of the present technology.
• FIG. 20 depicts the VEGF content in minced and digested placental composition of the present technology.
• Figure 21 depicts the VEGF content in minced and digested placental composition of the present technology after lysis in guanidine HCI.
• Figure 22 depicts the bFGF content in minced placental composition of the present technology after lysis in a tissue extraction buffer.
• FIG. 23 depicts the sustained growth factor release of minced and digested placental composition of the present technology.
• Figure 24 depicts the proliferative capacity of placental compositions of the present technology after isolation by digestion with Serva collagenase (24A) or Worthington collagenase (24B) after 14 days in culture.
• Figure 25 depicts the compatibility of minced placental composition with a variety of osteoconductive scaffolds.
• Figure 25A and B show placental compositions on HA-TCP- Collagen Foam and
• Figure 25C shows placental compositions on TransZgraft.
• Figure 26 depicts the tissue piece sizes from chorion minced with mezzaluna. Large square in upper left and right are 0.25 mm square (noted).
• FIG. 27A and B depict lipopolysaccharide (LPS) stimulated TNF-a released from various membrane preparations - Amnion+Chorion+Trophoblast (ACT), Chorion+Trophoblast (CT), Trophoblast (T), Amnion (AM), and Chorion (CM).
• LPS lipopolysaccharide
• FIG. 28 shows expression of IL-2Ra from T-cells stimulated by choriotrophoblast (CT) which secreted high levels of TNF-a.
• CT choriotrophoblast
• Choonic tissue or “Chorionic membrane” means the chorion or a portion thereof from placental tissue, e.g., the trophoblast, the somatic mesoderm, or combinations thereof.
• Amnionic tissue or “Amnionic membrane” means the amnion or a portion thereof from placental tissue, e.g., the epithelium layer; the basement membrane; the compact layer; the fibroblast layer; and the intermediate (spongy) layer.
• placental product and “placental composition” are used interchangeably and are the compositions described and claimed herein.
• "Placental composition” or “placental product” includes, but is not limited to, cells, extracellular matrix components, therapeutic factors, and tissue pieces/components containing placental cells, extracellular matrix components and/or therapeutic factors, and/or combinations thereof.
• the placental composition may also contain one or more cryopreservation agents.
• the placental product or placental compositions of the present technology can also be, for example, a graft, such as allografts or xenografts.
• Proportional dispersion means a composition or product formed by physical/ mechanical disruption of placental tissue.
• the placental dispersion can be formed from placental tissue from which a portion of the placental cells have been isolated and removed.
• the placental dispersion can be formed from placental tissue without isolating and removing placental cells.
• a dispersion may be in the form of a homogenate, a blend, a suspension, a colloid, or a solution, among others.
• Cellular Fraction refers to the portion of the digested placental tissue that remains after enzymatic digestion and after the tissue fraction is removed.
• the cellular fraction can comprise placental cells, extracellular matrix components, therapeutic factors, fragments and combinations thereof.
• tissue Fraction refers to the portion of the digested placental tissue that remains after enzymatic digestion after removal from the cellular fraction.
• the tissue fraction can comprise tissue fragments, including, cells, extracellular matrix components, therapeutic factors and combinations thereof.
• Placental tissue or “placental membrane” means tissue derived from the placenta in the broadest sense of the word. Placental tissue can be a whole placenta or any portion thereof. "Portions of the placenta” is meant to include chorion, amnion, a chorion and amniotic membrane (e.g., amnio-chorion), Wharton's jelly, umbilical cord, placental cotyledons, maternal decidua and/or combinations thereof. The placental tissue may be dissected or digested (or combinations thereof) to remove portions, membrane, or structures.
• Placental cells means any cell that can be obtained from a placenta, without regard to genetic origin (e.g., maternal vs. fetal), developmental origin (e.g., endodermal, ectodermal, or mesodermal), or differentiation.
• Placental cells may comprise any placental cells known in the art, for example, mesenchymal stem cells (MSCs), endometrial stromal cells (ESCs), placenta-derived mesenchymal progenitor cells, fibroblasts, epithelial cells, macrophages, and the like.
• MSCs mesenchymal stem cells
• ESCs endometrial stromal cells
• placenta-derived mesenchymal progenitor cells fibroblasts, epithelial cells, macrophages, and the like.
• Procental cells are further meant to require some feature of live cells such as one or more of metabolic activity, structural integrity (e.g., exclusion of a viability stain such as trypan blue), mitotic activity, signal transduction, and the like.
• Tissue injury means an injury of any tissue.
• Tissue injury can include, for example, injuries to tissues such as connective tissue (e.g., skin, cartilage, tendons, and/or ligaments, among others), bones, or other tissues or any organ.
• connective tissue e.g., skin, cartilage, tendons, and/or ligaments, among others
• bones or other tissues or any organ.
• tissue injury it is meant a pathology that involves or results from a mechanical insult, metabolic defect, disease or disorder, inflammation or other insult, defect, disease or disorder.
• tissue injuries include, but are not limited to burns, wounds (including tunnel wounds), ulcerations, and lacerations, ablations (including laser, freezing, cryo-surgery, heat and/or electrical ablations), and/or surgical incisions, among others.
• Placental compositions that are "depleted of immunogenicity,” or “depleted of immunogenic cells,” or “depleted of immunogenic factors,” or compositions that contain “depleted amounts of functional immunogenic cells” or “depleted amounts of one or more types of functional immunogenic cells” or compositions that are “depleted in amounts of functional immunogenicity” means one or more placental compositions of the present technology that retains live therapeutic cells and/or retains therapeutic efficacy for the treatment of tissue injury (or defect) yet is free, substantially free, or depleted of at least one immunogenic cell type (e.g., CD14+ macrophages, trophoblasts, and/or maternal blood cells) and/or immunogenic factor that is/are otherwise present in a native placenta, amniotic membrane or chorionic membrane.
• immunogenic cell type e.g., CD14+ macrophages, trophoblasts, and/or maternal blood cells
• a composition that is free, substantially free, or depleted of immunogenic cell types and/or immunogenic factors includes compositions that may retain some amount of immunogenic cells/factors but the retained amount is at a level that is insufficient to produce a functional response (e.g., below detectable amounts, in negligible amounts, in amounts insufficient to produce a functional immune response).
• MSC means mesenchymal stem cells and include fetal, neonatal, adult, or postnatal.
• MSCs include amniotic MSCs (AMSCs) and chorionic MSCs (CMSCs).
• MSCs generally express one or more of CD73, CD70, CD90, CD105, and CD166; and generally do not express CD45 and CD34.
• MSCs differentiate into mesodermal lineages (osteogenic, chondrogenic, and adipogenic).
• ECM Extracellular matrix
• tissue such as, for example, placental tissues including amniotic membrane, chorionic membrane, and/or chorioamniotic membrane.
• the term can include structural components of the ECM, such as collagens, laminins, fibronectin, hyaluronan, dermatan sulfate, heparin sulfate, chondroitin sulfate, decorin, and elastin, as well as soluble/functional therapeutic factors that may be present in the ECM (e.g., including proteins and fragments thereof).
• Native cells or "tissue cells” means cells that are native, resident, or endogenous to the placental membrane, i.e. cells that are not exogenously added to the placental membrane.
• Native factors means placental membrane factors that are native, resident, or endogenous to the placental membrane, i.e. factors that are not exogenously added to the placental membrane.
• Therapeutic cells or “beneficial cells” include cells and components present in the stromal layer, and/or the epithelial layer of the placenta and include, for example, MSCs, fibroblasts, and/or epithelial cells.
• Therapeutic factors means placenta-, chorionic membrane-, or amniotic membrane-derived factors that promote wound healing.
• Therapeutic factors also encompass molecules that may be classified as cell growth factors/proteins, tissue repair factors/proteins, as well as other factors and proteins that generally promote wound healing.
• Non-limiting examples of therapeutic factors include antimicrobial factors, chemoattractants, remodeling proteins such as proteases and protease inhibitors, immunoregulatory factors, chemokines, cytokines, growth factors, and other factors.
• Therapeutic factors also include factors that promote angiogenesis, cell proliferation, and epithelialization.
• Non-limiting examples of such factors include TGFa, TGFpi , TGFp2, TGFp3, EGF, HB-EGF, VEGF, VEGF-C, VEGF-D, HGF, PDGF-AA, PDGF-AB, PDGF-BB, PIGF, PEDF, Ang-2, IGF, IGFBP1 , IGFBP2, IGFBP3, adiponectin, a2-macroglobulin, FGFs (e.g., FGF-2/bFGF, KGF, KDG/FGF-7), matrix metalloproteinases (e.g., MMP-1 , MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-13), tissue inhibitors of metalloproteinases (e.g., TIMP1 , TIMP2), thrombospodins (e.g., TSP1 , TSP2), fibronectin, IL-1 RA, NGAL, defen
• Nemal cells refers to a mixed population of cells present (optionally in native proportions) composed of neonatal mesenchymal stem cells and neonatal fibroblasts. Both neonatal mesenchymal stem cells and neonatal fibroblasts are immunoprivileged; neither express surface proteins present on immunogenic cell types that trigger an immune response.
• 0087 "Stromal layer” refers to the layers in the placental membrane that do not contain the epithelial layer.
• 0088 “In vitro” describes the experiments and/or procedures performed outside of the living organism (e.g., under tissue culture conditions using artificial culture medium), including, but not limited to, culture expansion of cells.
• cryopreservation agent or “cryopreservative” or “cryoprotectant” are used interchangeably herein and are substances that help to prevent damage (e.g., cellular damage) during the freezing process.
• Suitable cryopreservation agents include, but are not limited to, Dimethyl Sulfoxide (DMSO), a glycerol, a glycol, a propylene glycol, an ethylene glycol, propanediol, polyethylene glycol (PEG), 1 ,2-propanediol (PROH) or a combination thereof.
• cryopreservation agents include, for example, one or more non-cell permeating cryopreservatives selected from, for example, polyvinyl pyrrolidione, a hydroxyethyl starch, a polysaccharide, a monosaccharide, an alginate, trehalose, raffinose, dextran, human serum albumin, ficoll, lipoproteins, polyvinyl pyrrolidone, hydroxyethyl strarch, autologous plasma or a combination thereof.
• cryopreservatives selected from, for example, polyvinyl pyrrolidione, a hydroxyethyl starch, a polysaccharide, a monosaccharide, an alginate, trehalose, raffinose, dextran, human serum albumin, ficoll, lipoproteins, polyvinyl pyrrolidone, hydroxyethyl strarch, autologous plasma or a combination thereof.
• cryopreservation solution refers to a composition comprising at least one cryopreservation agent.
• a cryopreservation solution or media may contain further components, for example, serum albumin, pharmaceutically acceptable carriers, buffers, electrolyte solutions, or saline (e.g., phosphate buffer saline).
• the cryopreservation solution or media may be a solution, a slurry, suspension, etc.
• the human amniotic membrane is the innermost of the fetal membranes deriving from the amniotic sac and constituting the lining of the amniotic cavity. It is approximately 0.02 to 0.5 mm thick.
• the AM consists of five layers: a single layer of epithelial cells rests on the basement membrane and contacts the amniotic fluid. An underlying layer of connective tissue is attached to the basement membrane. These connective tissues are comprised of three structural layers: a compact layer, a fibroblast layer (sometimes referred to as a mesenchymal layer), and a spongy layer. The spongy layer is adjacent to the cellular layer of the chorion.
• the amnion is essentially devoid of vasculature.
• CM human chorionic membrane
• placental compositions of the present technology can now be produced by mechanical/physical disruption or enzymatic digestion or a combination of both to produce a medicinal product of substantial and superior therapeutic value when administered to a whole or partial tissue injury or tissue defect.
• the placental composition(s) of the present technology also unexpectedly exhibited several advantageous properties.
• the technology described herein provides for placental compositions comprising manipulated placental tissues.
• the placental compositions can include cryopreserved amniotic membrane compositions, cryopreserved chorionic membrane compositions, and/or cryopreserved chorioamniotic membrane compositions.
• the cryopreservation methods retain high amounts of viable placental cells (i.e., cells that are native to the placental tissue(s)) and provide for the depletion of immunogenic cells and factors associated with immunogenic cells.
• compositions comprising cryopreserved disrupted amniotic, chorionic, and/or chorioamniotic membranes that comprise a combination of viable cells, therapeutic factors, extracellular matrix, and reduced immunogenicity, which find use in any number of beneficial therapeutic methods.
• the compositions can be applied to a wound or a tissue defect, and provide amounts of viable cells, therapeutic factors, extracellular matrix that can directly or indirectly induce a change in the region to which the membrane is applied (e.g., an adaptive medicine).
• compositions can provide for improved healing of wounds, such as chronic wounds or tunnel wounds by providing viable cells, therapeutic factors, and extracellular matrix in amounts that can provide or promote the normal stages of wound healing by any of promoting: (i) a reduction of the amount and/or activity of proinflammatory cytokines; (ii) an increase in the amount and/or activity of anti-inflammatory cytokines; (iii) a reduction of the amount and/or activity of reactive oxygen species; (iv) an increase in the amount and/or activity of anti-oxidant agents; (v) a reduction of the amount and/or activity of proteases; (vi) an increase in cell proliferation; (vii) an increase in angiogenesis; and/or (viii) an increase in cell migration to the wound.
• a chronic wound environment can include any one or more of 1 ) high levels of proinflammatory cytokines, 2) low levels of anti-inflammatory cytokines, 3) high levels of proteases and low levels of their inhibitors, as well as 4) high levels of oxidants and low levels of anti-oxidant to counter balance, the characteristics and functionality of the cryopreserved compositions disclosed herein are well suited to such applications.
• placental compositions of the present technology share certain properties of a fluid such as an ability to deform under an applied stress and can be quantified by measurements of viscosity.
• present placental composition can be spread over a surface(s) to which it is applied.
• one ml_ of placental composition can be spread topically to cover more than about 1 cm 2 , more than about 10 cm 2 , more than about 25 cm 2 , more than about 50 cm 2 , or more than about 100 cm 2 of skin.
• This fluid i.e., flowable property solves the problem of limited applicability of sheet-like products (e.g., skin grafts) to a variety of areas needing treatment (e.g., tunnel wounds, puncture wounds, large pressure wounds, curved surfaces, etc.). It also provides a means of rapid application.
• the placental composition can, for example, be injected or applied as an implant.
• the fluidity of the present placental compositions of the present technology now makes it practical for new uses such as application to tunnel wounds, articulating joints and curved surfaces.
• Placental cells may be obtained from any placental tissue (e.g., chorionic or amniotic). Placental cells may be obtained by processing placental tissue in any manner which retains cell viability of at least one cell type (e.g., MSCs). For example, placental cells may be isolated or purified from placental tissue (e.g., by collagenase digestion of the chorion) or may be obtained without isolation from one or more placental components (e.g., extracellular matrix).
• placental tissue e.g., chorionic or amniotic
• Placental cells may be obtained by processing placental tissue in any manner which retains cell viability of at least one cell type (e.g., MSCs).
• placental cells may be isolated or purified from placental tissue (e.g., by collagenase digestion of the chorion) or may be obtained without isolation from one or more placental components (e.g., extracellular matrix).
• Placental cells may be obtained by any method known in the art. Useful methods of obtaining placental cells (e.g., chorionic cells) are described, for example, by Portmann- Lanz et al. ("Placental mesenchymal stem cells as potential autologous graft for pre- and perinatal neuroregeneration"; American Journal of Obstetrics and Gynecology (2006) 194, 664-73), ("Isolation and characterization of mesenchymal cells from human fetal membranes”; Journal Of Tissue Engineering And Regenerative Medicine 2007; 1 : 296- 305.), and (Concise Review: Isolation and Characterization of Cells from Human Term Placenta: Outcome of the First International Workshop on Placenta Derived Stem Cells").
• placental cells e.g., chorionic cells
• placental cells are obtained by contacting placental tissue with one or more digestive enzymes, for example, by immersing placental tissue (e.g., a chorion, or placental tissue lacking trophoblasts) in a solution containing the digestive enzyme.
• the digestive enzyme may be any digestive enzyme known in the art.
• the digestive enzyme may also be a combination of enzymes.
• Exemplary digestive enzymes include one or more: collagenases (e.g., collagenase type I, II, III and IV), matrix metalloprotease, neutral proteases, papains, deoxyribonucleases, serine protease (e.g., trypsin, chymotrypsin, elastase), or any combination thereof).
• collagenases e.g., collagenase type I, II, III and IV
• matrix metalloprotease e.g., matrix metalloprotease, neutral proteases, papains, deoxyribonucleases, serine protease (e.g., trypsin, chymotrypsin, elastase), or any combination thereof).
• placental cells are obtained from a chorion by contacting a chorion (e.g., a chorion lacking trophoblasts) with a collagenase (e.g., collagenase type II).
• a collagenase e.g., collagenase type II
• the collagenase may be present in any suitable concentration, for example, about 10 U/mL to about 1000 U/mL, and in any suitable collagenase solvent, such as DMEM, and at any suitable temperature, for example 37 °C.
• the chorion may be contacted with the digestive enzyme for any suitable period of time.
• the chorion is contacted with a collagenase (e.g., collagenase type II) for less than about any of: about 16 hours, about 12 hours, about 8 hours, about 3 hrs, about 2 hr, or about 1 hr.
• the chorion is contacted with the collagenase (e.g., collagenase type II) for less than about 1 hour, for example, less than about any of: about 60 min, about 50 min, about 40 min, about 30 min, about 20 min, about 15 min, about 10 min, or about 5 min.
• the chorion is contacted with a collagenase for a limited period of time such that a substantial portion of the placental tissue is retained on a 100 micron filter.
• the chorion is disrupted to form a dispersion and the population of cells is combined with (e.g., added to) the dispersion.
• the placental tissue is disrupted, for example by mincing, without contacting the placental tissue with a collagenase or similar digestive enzyme. Disrupting the placental tissue without a digestion step is less time consuming than a process that includes a digestion step and provides a minimally manipulated placental composition with a high viability of live cells after cryopreservation and subsequent thawing.
• the placental compositions prepared in accordance with the present technology provide a therapeutically effective amount of viable cells without the need for ex vivo expansion of the placental cells.
• ex vivo expansion is a known method of increasing the number of viable cells in a population, such a step often leads to changes in the population make-up or distribution of cell phenotype.
• various cells in a population may expand at different rates and expansion may also induce differentiation.
• at least one embodiment of the present technology provides a placental composition comprising placental cells derived from a placental tissue wherein the placental cells exhibits a phenotypic distribution of cells which is substantially similar to the cells of the placental tissue of origin.
• a placental dispersion may be provided by disrupting a placenta (e.g., a chorion).
• the disruption of placental tissue may be accomplished by any physical/mechanical method of disrupting tissue (i.e. use of a "tissue disruptor” or “means for disruption”).
• disruption may comprise homogenization, maceration, use of a blender, crushing, or mincing, among others.
• Disruption may additionally or alternatively comprise shearing, dicing, or chopping.
• Disruption may additionally or alternatively comprise sonication.
• the placental tissue may be disrupted for any suitable duration which produces a dispersion from the placenta.
• the placenta may be disrupted (e.g., homogenized) for less than about 1 minute, about 30 seconds, about 20 sec, about 15 sec, about 10 sec, or about 5 seconds.
• the placenta may be disrupted by mincing for at least about one minute, for at least about 5 minutes, for at least about 15 minutes.
• the placental tissue of the present technology can be disrupted by mincing the placental tissue into approximately uniformly sized pieces to form the placental dispersion.
• the mincing step can be conveniently accomplished by using a tool having one or more blades effective for cutting the placental tissue into sufficiently small sized pieces to form the dispersion.
• suitable tools for mincing the placental tissue include but are not limited to knives, scalpels, herb mincers, or a mezzaluna.
• the minced tissue pieces are triturated (pipetted up and down through a pipette) to evenly distribute the placental pieces in the placental dispersion.
• the present technology provides one or more placental compositions that comprise minced placental tissue wherein the average size for the tissue pieces is from about 0.42 mm 2 to about 1 .137 mm 2 . In some aspects, the average size is about at least 0.42 mm 2 . In other aspects, the average size is about 100 ⁇ 2 to about 25 mm 2 .
• the average sizes of the tissue pieces are able to still pass through a syringe. In some aspects, the average sizes of the tissue pieces can pass through an 18 gauge needle or larger. Other syringe gauges are also envisaged.
• placental tissue can be disrupted sufficiently to form a placental composition with fluid characteristic and yet retain viable cells.
• live cells in the placental compositions of the present technology can additionally comprise placental cells that are derived from the placental dispersion.
• a placental composition of the present technology may comprise one or more therapeutic factors where the therapeutic factors are components of the placental dispersion or components released into the placental composition by the placental cells or a combination thereof.
• 00110 It has surprisingly been discovered that the content of therapeutic factors in placental compositions made according to the present technology have an unexpected therapeutic value. Such content of therapeutic factors as taught herein is accordingly referred to here as a "therapeutic profile" and can be provided by the cells, therapeutic factors and extracellular matrix components or combinations thereof.
• a therapeutic profile is one that provides two or more, or three or more, or four or more or greater therapeutic factors listed in Table 1 , Table 2, and Table 5.
• the therapeutic factors are present in an amount of about 20% to about 500% of the mean concentration set forth in Table 1 , Table 2 or Table 5.
• the therapeutic factors are present in an amount of about 20% to about 500% of the minimum and the maximum (respectively) of the values set forth in Table 1 , Table 2 or Table 5.
• therapeutic factors can be placental-derived factors such as angiogenic factors, chemokines, cytokines, growth factors, matrix metalloproteases, proteases, protease inhibitor, and combinations thereof, among others.
• the present placental compositions can comprise any of these therapeutic factors.
• useful placental compositions of the present technology can have a therapeutic profile as set forth in Table 1 , Table 2, or Table5, and can have a therapeutic profile comprising for example, at least two or more therapeutic factors, at least three or more therapeutic factors, or at least four or more therapeutic factors or greater as noted above.
• the compositions can further comprise at least one extracellular matrix component or combinations of extracellular matrix components.
• compositions can optionally comprise a therapeutic profile of one or more of a PDGF (e.g., PDGF-BB), EGF, FGF, TGF- ⁇ , TGF- ⁇ 3, and VEGF and/or one or more of IL-8, IL-6, and MCP-1 .
• a PDGF e.g., PDGF-BB
• EGF EGF
• FGF FGF
• TGF- ⁇ TGF- ⁇
• TGF- ⁇ 3 VEGF
• IL-8 interleukin-6
• MCP-1 MCP-1
• Placental compositions of the present technology can comprise a therapeutic profile of one or more therapeutic factors which promote, for example, the migration of cells into the wound area (e.g., HGF and/or KGF), optionally in combination with a growth factor such as TGF- ⁇ .
• Suitable cells that migrate into the wound area include, but are not limited to, epithelial cells, endothelial cells, fibroblasts, MSC, or combinations thereof.
• the concentration of such therapeutic factors is about 25% of the minimum values set forth in Table 1 .
• the placental compositions of the present technology can comprise a therapeutic profile of one or more therapeutic factors which provide antiinflammatory cytokines that may restart, stimulate, or enhance, for example, the healing process, for example, IL-10 and PGE-2.
• Placental compositions of the present technology can comprise a therapeutic profile of therapeutic factors that are mitotic or growth promoting as well.
• Placental compositions of the present technology can contain HGF and KGF.
• Placental compositions of the present technology can also comprise a therapeutic profile of therapeutic factors comprising one or more angiogenic factors (e.g., VEGF and/or bFGF) and can optionally additionally comprise one or more growth factors (e.g., TGF- ⁇ and/or TGF-32).
• angiogenic factors e.g., VEGF and/or bFGF
• growth factors e.g., TGF- ⁇ and/or TGF-32).
• Exemplary placental compositions of the present technology contain a therapeutic profile of VEGF levels greater than about 10 pg/mL or greater than about 50 pg/mL or greater than about 100 pg/mL.
• an exemplary placental product can comprise greater than about 200 pg/mL of VEGF as detailed in Example 9.
• the placental product of the present technology contains a therapeutic profile of VEGF levels greater than about 1000 pg/mL or greater than about 2000 pg/mL, for example, about 2000 pg/mL to about 3000 pg/mL.
• Exemplary placental compositions of the present technology contain a therapeutic profile of bFGF levels greater than any of about 10 or 100 or 1 ,000 or 10,000 pg/mL.
• An exemplary placental product can comprise greater than about 1 1 ,000 pg/mL of bFGF.
• Suitable amounts of VEGF and/or bFGF are exemplified in Example 9, 26, 27 and 28.
• such bFGF-comprising placental compositions of the present technology are useful for burn wound healing.
• Placental compositions of the present technology can comprise a therapeutic profile of TGF- ⁇ , TGF-32, and/or TGF-33.
• An exemplary placental composition comprises bFGF, TGF- ⁇ , TGF-32, and TGF-33.
• placental compositions of the present technology are useful when the skin pathology being treated involves an inflammatory or a scaring pathology.
• the placental product produces TGF- ⁇ AND TGF-33 in ratios effective to reduce or prevent formation of scar tissue.
• Placental compositions of the present technology may comprise a therapeutic profile of one or more protease inhibitors, such as tissue inhibitors of matrix metalloproteinases (TIMPs), alpha-2 macroglobulin, and/or thrombospondins.
• TGFPs tissue inhibitors of matrix metalloproteinases
• a placental composition (e.g., derived from chorion) comprises one or more protease inhibitors.
• a placental composition (e.g., derived from chorion) comprises one or more protease inhibitors and extracellular matrix proteins.
• a placental composition (e.g., derived from chorion) comprises one or more protease inhibitors and viable cells.
• a placental composition (e.g., derived from chorion) comprises one or more protease inhibitors, extracellular matrix proteins, and viable cells.
• the present inventors believe that the placental compositions of the present technology have enhanced efficacy compared to non-living wound healing products because the placental cells and the therapeutic factors interact with or adapt to the host environment. Such interaction or adaptation stimulates the release of therapeutic factors or changes in the placental factor profile over time after administration, resulting in a dynamic therapy that is effective in all phases of wound repair unlike conventional wound and/or non-dynamic wound therapies.
• such placental compositions of the present technology can optionally maintain surprising integrity for extended periods of time resulting in placental compositions that require less frequent applications and superior treatment of tissue injuries (or defects) such as, for example, burns and wounds, among others.
• tissue injuries or defects
• the growth factors in such placental compositions can demonstrate a longer half-life in comparison to other growth factor therapies such as Amnion-derived Cellular Cytokine Solution (ACCS).
• ACCS Amnion-derived Cellular Cytokine Solution
• compositions of the present technology can be administered as a dermatologically acceptable pharmaceutical product.
• active pharmaceutical ingredients or excipients or combinations thereof can be added with or thereto or combined with or thereto.
• Viscosity values that are useful and desirable according to the present technology vary as a function of the indication being treated. For example, where broad coverage (i.e. large areas of skin) or lower concentrations of placental compositions are desired, a less viscous formulation is advantageous. Examples of less viscous formulations are those of about 1 ,000 cP to about 50,000 cP, or about 2,000 cP to about 25,000 cP, or about 2,000 cP to about 10,000 cP, or about 5,000 cP to about 15,000 cP. Such less viscous compositions can facilitate spreading of the applied placental composition(s) of the present technology.
• a more viscous formulation is advantageous.
• examples of more viscous formulations are about 20,000 cP to about 200,000 cP or about 50,000 cP to about 100,000 cP.
• the desired viscosity can be attained according to the present technology by adjustments of the dispersion method (discussed elsewhere herein) or by selection of a carrier, such as saline or a dermatologically acceptable thickening agent and empirically determining the concentration necessary to achieve a desired viscosity or flow characteristic.
• a carrier such as saline or a dermatologically acceptable thickening agent
• the compositions can be formulated into a liquid, a solution, a gel, a slurry, or suspension, among others.
• the placental compositions of the present technology can optionally include one or more antibiotics, emollients, keratolytics agents, humectants, anti-oxidants, preservatives, or combinations thereof. Other additives are also envisaged.
• a placental composition comprises albumin, such as human serum albumin (HSA) or bovine serum albumin (BSA).
• albumin such as human serum albumin (HSA) or bovine serum albumin (BSA).
• the placental composition comprises an electrolyte solution, for example, to provide physiological osmolality and pH (e.g., Plasma-Lyte A).
• the placental composition comprises a cryopreservation agent, such as DMSO, glycerol, glycerin, sugars, or a mixture thereof.
• a placental composition comprises from about 3% to about 100% by volume of at least one cryopreservation agent in the final composition, preferably about 3% to about 90%, alternatively from about 5% to about 50%, alternatively from about 5% to about 20%, alternatively from about 3% to about 10% of at least one cryopreservation agent by volume, for example, DMSO.
• the placental composition comprises about 5% to about 20% of a cryopreservation agent and about 0% to about 15 % albumin by volume.
• the placental composition may further comprise at least one pharmaceutically acceptable carrier, for example, saline or an electrolyte solution.
• a placental composition comprises albumin, an electrolyte solution, and a cryopreservation agent.
• the therapeutic composition comprises about 1 % to about 15% albumin by volume and about 5% to about 20% cryopreservation agent by volume (e.g., about 10%) in the final composition.
• the albumin is HSA
• the electrolyte solution is Plasma-Lyte A
• the cryopreservation agent is DMSO.
• the placental composition comprises the cryopreservation agent in an amount of about 3% to about 100% by volume of the final composition, more preferably about 3% to about 20% by volume of the final composition.
• a placental composition of the present technology may be manufactured from a placenta in any suitable manner that provides the technical features taught herein. Any placental tissue is useful according to the present technology. Some of the embodiments of the present technology set forth here are meant to specifically embrace placental compositions where the placental dispersion is a dispersion of chorion that is depleted of or lacking trophoblastic components. Alternatives are also envisaged.
• the placental dispersion and the placental cells are derived from a different placenta or different placental portion (e.g., parallel processing). It will also be appreciated that the dispersion can include a therapeutic (e.g., drug or biologic). In another, the placental dispersion and the placental cells are derived from the same placenta or the same placental portion (e.g., sequential processing).
• the placental composition is manufactured by the following steps comprising:
• placental cells obtained from the digested placental tissue;
• tissue disruptor disrupting the digested placental tissue with a tissue disruptor to form a placental dispersion comprising therapeutic factors, extracellular matrix, placental cells, and tissue pieces;
• the disrupting step comprises homogenizing the digested placental tissue. In other aspects, the disrupting step comprises mincing the digested placental tissue.
• a placental composition is manufactured by the steps comprising:
• a first placental e.g., chorionic or amniotic
• a placental composition is manufactured by the steps comprising:
• a process for preparing a placental tissue composition according to the present technology comprises the steps of:
• a providing a placental tissue comprising placental cells, therapeutic factors, and extracellular matrix components; b. disrupting at least a portion of the placental tissue to form a placental dispersion comprising placental tissue pieces, placental cells, therapeutic factors and extracellular matrix components;
• cryopreserving the placental dispersion to form a placental tissue composition wherein after cryopreservation and subsequent thawing of the placental tissue composition, greater than 40% of the placental cells are viable and wherein the composition is depleted in functional immunogenic cells.
• a process for preparing a placental tissue composition according to the present technology comprises the steps of:
• cryopreserving the placental tissue composition wherein after cryopreservation and subsequent thawing of the placental tissue composition, greater than 40% of the placental cells are viable and wherein the composition is depleted in functional immunogenic cells.
• At least 70% of the placental cells are viable, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%. It should be appreciated by those skilled in the art that such a viability percentage is non-exhaustive and includes increments in between and greater than the percentages presented.
• the methods of the present technology further comprise the step of cryopreserving the placental compositions.
• the method of cryopreserving can include adding at least one cryopreservation agent to the composition, placing the composition at a temperature range of about 2°C to about 8°C for about 3 minutes to about 240 minutes, for example from 10 minutes to about 60 minutes; and then subsequently freezing the composition at a temperature range of about -20° C to about -196°C (alternatively about -45 °C to about -80 °C).
• the placental tissue can be a chorion tissue such as a chorion tissue that has been processed to reduce the number of trophoblastic cells.
• compositions of the present technology can be manufactured or provided with a bandage or wound dressing.
• the placental composition is immunocompatible. Immunocompatability can be accomplished by any selective depletion step that removes immunogenic cells or factors or immunogenicity from the placenta or placental derived tissue (or amniotic membrane thereof).
• the placental composition is made immunocompatible by selectively depleting it of functional immunogenic cells.
• a placenta can be made immunocompatible by selectively reducing or removing immunogenic cells from the placenta (or amniotic membrane thereof) relative to therapeutic cells.
• immunogenic cells can be removed by killing the immunogenic cells or by purification of the placenta therefrom.
• the placental composition is made immunocompatible by selectively depleting trophoblasts, for example, by removal of the trophoblast layer.
• the placental composition is made immunocompatible by selective depletion of functional CD14+ macrophages, optionally as demonstrated by a substantial decrease in lipopolysaccharide (LPS) stimulation of TNFa release or by mixed lymphocyte reaction (MLR) assay.
• LPS lipopolysaccharide
• MLR mixed lymphocyte reaction
• the placental composition is made immunocompatible by selective depletion of maternal blood cells.
• the placental composition is made immunocompatible by selective depletion of functional CD14+ macrophages, trophoblasts, and/or maternal blood cells.
• the placental composition is made immunocompatible by selective depletion of trophoblasts and/or CD14+ macrophages, optionally as demonstrated by a substantial decrease in LPS stimulation of TNFa release or by MLR assay.
• the depleted amounts of functional immunogenic cells produce immunogenic factors in amounts that are below levels sufficient to produce an immune response. In some embodiments, the depleted amounts of functional immunogenic cells produce immunogenic factors in amounts below detectable limits.
• trophoblasts are depleted or substantially removed to produce the placental tissue from which the placental cells or the placental dispersion or both are derived.
• a placental composition has one or more of the following superior features (among others):
• a. is substantially non-immunogenic
• c. provides enhanced therapeutic efficacy.
• Trophoblasts may be removed in any suitable manner which substantially diminishes the trophoblast content of the placental composition.
• the trophoblasts are selectively removed.
• the trophoblasts are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the chorionic membrane (e.g., MSCs, therapeutic factors, extracellular matrix, etc).
• the trophoblasts are removed before isolating a population of cells and/or disrupting the placental tissue.
• One method of removing trophoblasts comprises treating the placenta (e.g., chorion or amnio-chorion) with a digestive enzyme such as dispase for about 30 to about 45 minutes and separating the trophoblasts from the placenta.
• the step of separating comprises mechanical separation such as scraping.
• a suitable method of scraping comprises scraping with a soft instrument such as a finger.
• trophoblasts are removed while retaining the basement layer, reticular layer, and/or stromal cell layer of the amniotic membrane.
• trophoblasts are removed before cryopreservation.
• a placental composition has one or more of the following superior features:
• Functional macrophages can be removed in any suitable manner which substantially diminishes the macrophage content of the placental composition.
• the macrophages are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the placenta (e.g., MSCs, therapeutic factors, extracellular matrix components, etc).
• a majority (e.g., substantially all) of the macrophages are removed.
• Macrophages include, but are not limited to, CD14+, CD1 1 b+, CD18+, CD40+ and CD86+.
• One method of removing immune cells such as macrophages comprises killing the immune cells by rapid freezing rates such as 60-100°C/min.
• Another method of removing immune cells comprises killing the immune cells by holding the cells at temperatures (e.g., about 2°C to about 8°C, e.g., "refrigerator” temperatures) for a period of time, and then freezing the immune cells at a rate of about 1 °C/min.
• stromal cells e.g., MSCs
• the present inventors have discovered a method of whereby CD14+ macrophages can be selectively killed by placing the placenta for a period of time (e.g., for at least about 3 minutes to about 240 minutes, for example, for about 10 to about 60 mins) at a temperature above freezing (e.g., incubating at about 2 °C to about 8°C) and then freezing the placenta (e.g., incubating at about -20°C to about -196°C, e.g., about -80°C ⁇ 5°C).
• a period of time e.g., for at least about 3 minutes to about 240 minutes, for example, for about 10 to about 60 mins
• a temperature above freezing e.g., incubating at about 2 °C to about 8°C
• freezing the placenta e.g., incubating at about -20°C to about -196°C, e
• the step of freezing comprises freezing at a rate of less than 10°C/min (e.g., less than about 5°C/min such as at about 1 °C/min).
• the step of refrigerating comprises incubating the composition containing at least one cryopreservation agent (e.g., DMSO) for a period of time sufficient to allow the cryopreservation agent to penetrate (e.g., equilibrate with) the placental tissues.
• the composition further comprises albumin and optionally a pharmaceutically acceptable carrier, e.g., saline or electrolyte solution.
• the step of freezing comprises reducing the temperature at a rate of about 1 °C/min.
• the step of freezing comprises freezing at a rate of less than 10°C/min (e.g., less than about 5°C/min such as at about 1 °C/min).
• the step of incubating the composition containing at least one cryopreservation agent e.g., DMSO
• at least one cryopreservation agent e.g., DMSO
• a temperature of about -10°C to about 15°C e.g., at about 2°C to about 8°C
• at least about 3 minutes to about 240 minutes for example, about any of: 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 100 min, 120 min, 180 min, 240 min.
• the step of incubating the composition containing at least one cryopreservation agent comprises placing the composition at a temperature of about -10°C to about 15°C (e.g., at about 2°C to about 8°C) for about any of: 10-120min, 20-90 min, or 30-60 min, 10-240 min.
• the step of freezing comprises freezing at a rate of less than 10°C/min (e.g., less than about 5°C/min such as at about 1 °C/min).
• maternal blood cells are depleted or removed from the placental composition.
• a placental composition has one or more of the following superior features:
• Maternal blood cells can be removed in any suitable manner which substantially diminishes such cell content of the placental composition.
• the maternal blood cells are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the placenta (e.g., therapeutic cells (e.g., MSCs), therapeutic factors, anti-oxidant agents, anti-inflammatory agents, etc).
• therapeutic cells e.g., MSCs
• therapeutic factors e.g., anti-oxidant agents, anti-inflammatory agents, etc.
• removal of maternal blood cells comprises rinsing the amniotic membrane (e.g., with buffer such as PBS) to remove gross blood clots and any excess blood cells.
• removal of maternal blood cells comprises treating the amniotic membrane with an anticoagulant (e.g., citrate dextrose solution).
• an anticoagulant e.g., citrate dextrose solution
• removal of maternal blood cells comprises rinsing the amniotic membrane (e.g., with buffer such as PBS or D-PBS) to remove gross blood clots and any excess blood cells, and treating the amniotic membrane with an anticoagulant (e.g., citrate dextrose solution).
• an anticoagulant e.g., citrate dextrose solution
• the chorionic membrane is retained and removal of maternal blood cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord. The chorion on the umbilical side of the placenta is not removed due to the vascularization on this side.
• the chorionic membrane is retained and removal of maternal blood cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord and rinsing the amniotic membrane and chorionic membrane (e.g., with buffer such as PBS or D-PBS) to remove gross blood clots and any excess blood cells.
• buffer such as PBS or D-PBS
• the chorionic membrane is retained and removal of maternal blood cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord and treating the amniotic membrane and chorionic membrane with an anticoagulant (e.g., citrate dextrose solution).
• an anticoagulant e.g., citrate dextrose solution
• the chorionic membrane is retained and removal of maternal blood cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord, rinsing the chorionic membrane amniotic membrane (e.g., with buffer such as PBS) to remove gross blood clots and any excess blood cells, and treating the amniotic membrane with an anticoagulant (e.g., citrate dextrose solution).
• an anticoagulant e.g., citrate dextrose solution
• the placental composition is selectively depleted of immunogenicity as demonstrated by a reduction in LPS stimulated TNF-a release. In one embodiment, the placental composition is selectively depleted of macrophages.
• TNF-a is depleted by killing or removal of macrophages.
• the level of TNF- a is less than about 350 pg/cm 2 , alternatively less than about 225 pg/cm 2 , alternatively less than about 100 pg/cm 2 or alternatively less than about 70 pg/cm 2 or less.
• TNF- a is inhibited at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.
• Immunocompatibility can be demonstrated by any means commonly known by the skilled artisan, such demonstration can be performed by the mixed Lymphocyte reaction (MLR) and by lipopolysaccharide (LPS)-induced Tumor Necrosis Factor (TNF)-a secretion.
• MLR mixed Lymphocyte reaction
• LPS lipopolysaccharide
• TNF Tumor Necrosis Factor
• a placental composition of the present technology may be used fresh or may be preserved for a period of time.
• the placental composition is cryopreserved.
• the placental composition is placed in a pharmaceutically acceptable container that can withstand cryopreservation temperatures, with at least one cryopreservation agent and cryopreserved by freezing (e.g., from about -20 °C to about -196°C, such as about -80 °C).
• Suitable containers for cryopreservation of the placental composition are biocompatible, non- immunogenic and able to withstand temperatures of about -196°C to about 205 °C and include vials, pouches, bottles, and bags.
• the placental composition (in its container) may be frozen in a Styrofoam box to control the freezing rate.
• Freezing may comprise storage in a cryopreservation medium containing at least one cryopreservation agent, include, but are not limited to, for example, DMSO, glycerol, sugars, or mixtures thereof. Freezing may comprise, for example, incubating the placental composition at a temperature of about 2°C to about 8°C, for example at about 4°C, for about 3 minutes to about 240 minutes, for example, 10 min to 60 min or 30-60 min, and then incubating at about -45 °C to about - 196°C, for example, -80°C until use. The placental composition may then be thawed for use.
• the placental compositions of the present technology retain a high amount of cell viability after cryopreservation and subsequent thawing. In some aspects, after thawing, at least 40% of the placental cells are viable. In some other aspects, the placental compositions retain at least about 70% viability, alternatively at least about 75% viability, alternatively about 80% viability, alternatively about 85% viability, alternatively about 90% viability, alternatively about 100% viability.
• a placental composition may be formulated to include at least one cryopreservation agent before cryopreservation.
• cryopreservation agents include
• compositions may further be formulated with additional components such as albumin (e.g., HSA or BSA), an electrolyte solution (e.g., Plasma-Lyte
• the placental compositions comprises 0% to 15% albumin by volume and about 3% to about 100%, more suitably 3% to about 50% of at least one cryopreservation agent by volume, for example, about 1 % to about 15% albumin by volume and about 5% to about 100% cryopreservation agent by volume (e.g., about 5% to about 10%).
• a placental composition can be formed by the addition of cryopreserved placental cells of the present technology to a fresh (never frozen) placental dispersion or to a frozen placental dispersion or to a lyophilized placental dispersion.
• a placental composition can be formed by the addition of fresh placental cells of the present technology to a frozen placental dispersion or to a lyophilized placental dispersion.
• Cryopreserved placental compositions may be thawed before use. Suitable methods of thawing would be understood by one skilled in the relevant art.
• the cryopreserved placental compositions may be thawed at room temperature or at about 37 °C.
• the cryopreserved placental compositions are thawed at a sufficiently fast rate as to retain high viability of cells (e.g., at least 40% viable cells, more preferably at least 70% viable cells).
• a 0.3 mL cryopreserved placental composition may be thawed in less than a minute at room temperature (about 25 °C) while a 1 mL cryopreserved placental composition may be thawed at about 3 minutes at room temperature. It would be understood by one skilled in the relevant art that increasing the volume of the placental product will increase the thaw time. Further, it would be understood that the thaw time may be reduced by thawing the composition at a higher temperature (e.g., about 37°C).
• the cryopreserved compositions have a surprisingly long shelf-life or stability to retain viable cells when frozen for extended periods of time.
• the cryopreserved products may be store at about -20 °C to about -196°C (e.g., about -45 °C to about -80 °C) for two years or more with retention of high viability (at least 70% retention of viable cells) once thawed.
• cryopreserved compositions can be stored at about -20 °C to about -196°C (e.g., about -45 °C to about -80°C) for at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 15 months, at least about 24 months, at least about 36 months before thawing with a high retention of viable cells (e.g., at least 40% viable cells, alternatively at least 50% viable cells, alternatively at least about 70% viable cells, alternatively at least about 80%, about 85%, 90%, or 95% viable cells).
• viable cells e.g., at least 40% viable cells, alternatively at least 50% viable cells, alternatively at least about 70% viable cells, alternatively at least about 80%, about 85%, 90%, or 95% viable cells.
• the placental compositions, and particularly the cryopreserved compositions described herein provide an amount of viable cells, therapeutic factors, and extracellular matrix components that are effective to promote a number of beneficial therapeutic activities and effects.
• the compositions may be applied and provide amounts of therapeutic factors, viable cells, and extracellular matrix and provide an environment that can promote endogenous cells to produce any number of therapeutic factors that provide the same or similar therapeutic benefit.
• the placental compositions of the present technology may be used to treat any tissue injury.
• a method of treatment may be provided, for example, by administering to a subject in need thereof, a placental composition of the present technology.
• the placental compositions of the present technology may also be used to regenerate tissue, directly or indirectly.
• An administration method of the present technology is topical administration.
• Administering the present technology can also involve administration to an internal tissue where access is gained by a surgical procedure.
• the placental composition can be injected through a syringe or needle.
• Placental compositions are autologous, allogeneic, or xenogeneic.
• a placental composition is administered to a subject to treat a wound or tissue defect.
• the wound is a laceration, scrape, thermal or chemical burn, incision, puncture, or wound caused by a projectile.
• the wound is an epidermal wound, skin wound, chronic wound, acute wound, external wound, internal wounds, ocular wounds, congenital wound, ulcer, or pressure ulcer.
• the wound may be tunnel wounds. Tunnel wounds may be caused by, for example, but not limited to infection, prolonged inflammation for chronic wounds, pressure and shear forces that are concentrated where tissue layers meet, inadequate drainage absorption due to insufficient wound packing and degradation of newly granulated tissue due to too much wound packing. Such wounds may be accidental or deliberate, e.g., wounds caused during or as an adjunct to a surgical procedure.
• the wound is closed surgically prior to administration.
• the injury is a burn, such as a first-degree burn, second- degree burn (partial thickness burns), third degree burn (full thickness burns), infection of burn wound, infection of excised and unexcised burn wound, loss of epithelium from a previously grafted or healed burn, or burn wound impetigo.
• the injury is an ulcer, for example, a pressure ulcer, a diabetic ulcer, venous skin ulcers, or foot or leg ulcers.
• a placental composition is administered by applying the placental composition directly over the skin of the subject, e.g., on the stratum corneum, on the site of the wound, so that the wound is covered.
• the placental composition is covered with a non-adhesive dressing.
• the placental composition may be administered as an implant, e.g., as a subcutaneous implant.
• a placental composition is administered to the epidermis to reduce rhytids or other features of aging skin.
• Such treatment is also usefully combined with so-called cosmetic surgery (e.g., rhinoplasty, rhytidectomy, hair restoration, etc.).
• a placental composition is administered to the epidermis to accelerate healing associated with a dermal ablation procedure or a dermal abrasion procedure (e.g., including laser ablation, thermal ablation, electric ablation, deep dermal ablation, sub-dermal ablation, fractional ablation, and microdermal abrasion).
• a dermal ablation procedure or a dermal abrasion procedure e.g., including laser ablation, thermal ablation, electric ablation, deep dermal ablation, sub-dermal ablation, fractional ablation, and microdermal abrasion.
• traumatic wounds e.g., civilian and military wounds
• surgical scars and wounds spinal cord injury
• avascular necrosis ablations
• ischemia e.g., ischemia
• a placental composition of the present technology is used in a tissue graft procedure.
• the placental composition is applied to a portion of the graft which is then attached to a biological substrate (e.g., to promote healing and/or attachment to the substrate).
• tissue such as skin, cartilage, ligament, tendon, periosteum, perichondrium, pericardium, synovium, fascia, mesenter and sinew can be used as tissue graft (e.g., any natural or synthetic grafts which are biocompatible).
• a placental composition is used in a tendon or ligament surgery to promote healing of a tendon or ligament.
• the placental composition is applied to a portion of a tendon or ligament which is attached to a bone.
• the surgery can be any tendon or ligament surgery, including, e.g., knee surgery, shoulder, leg surgery, arm surgery, elbow surgery, finger surgery, hand surgery, wrist surgery, toe surgery, foot surgery, ankle surgery, and the like.
• the placental composition can be applied to a tendon or ligament in a grafting or reconstruction procedure to promote fixation of the tendon or ligament to a bone.
• placental compositions of the present technology provide superior treatment (e.g., healing, healing time and/or healing strength) for wounds and defects of any tissue, cartilage and bone.
• tissue is connective tissue or nerve tissue.
• the placental compositions are used to heal connective tissues such as tendons and ligaments. Tendon and ligament surgeries can involve the fixation of the tendon or ligament to bone. Without being bound by theory, the present inventors believe that osteogenic and/or chondrogenic potential of MSCs in the present placental compositions promotes the healing process and healing strength of bone or cartilage.
• the placental composition may treat any type of tissue (e.g., bone, ligament, tendon, cartilage or soft tissue) wounds and injuries or any type of defects.
• tissue e.g., bone, ligament, tendon, cartilage or soft tissue
• the present placental compositions provide an alternative or adjunctive treatment to periosteum-based therapies.
• useful periosteum based treatments are described in Chen et al. ("Enveloping the tendon graft with periosteum to enhance tendon-bone healing in a bone tunnel: A biomechanical and histologic study in rabbits"; Arthroscopy. 2003 Mar;19(3):290-6), Chen et al. (“Enveloping of periosteum on the hamstring tendon graft in anterior cruciate ligament reconstruction"; Arthroscopy.
• the placental composition is injected into the area surrounding the tendon or ligament.
• the tendon is placed into a bone tunnel before it is attached to the bone.
• the tendon or ligament surgery is a graft procedure, wherein the placental composition is applied to the graft.
• the graft is an allograft, xenograft, or an autologous graft.
• the tendon or ligament surgery is repair of a torn ligament or tendon, wherein the placental composition is applied to the torn ligament or tendon.
• Non-limiting examples of tendons to which a placental composition can be applied include a digitorum extensor tendon, a hamstring tendon, a bicep tendon, an Achilles Tendon, an extensor tendon, and a rotator cuff tendon.
• a placental composition of the present technology is used to reduce fibrosis by applying the placental composition to a wound site.
• the disclosure provides a method of promoting tissue repair and/or tissue regeneration in a subject comprising administering to the subject a composition as disclosed herein wherein the administration provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to promote tissue repair and/or tissue regeneration.
• the method is used in combination with a surgical procedure selected from the group consisting of a tissue graft procedure, tendon surgery, ligament surgery, bone surgery, and spinal surgery.
• the tissue is human tissue.
• the human tissue is cartilage, skin, ligament, tendon, or bone.
• the compositions may directly or indirectly stimulates tissue regeneration.
• a placental composition of the present technology is used as an anti-adhesion wound barrier, wherein the placental composition is applied to a wound site, for example, to reduce fibrosis (e.g., post-operative fibrosis).
• fibrosis e.g., post-operative fibrosis
• Non-limiting examples of wound sites to which the placental composition can be applied include those that are surgically induced or associated with surgery involving the spine (e.g., spinal fusions), reconstructive surgery, laminectomy, knee, shoulder, or child birth, trauma related wounds or injuries, cardiovascular procedures, angiogenesis stimulation, brain/neurological procedures, hernia repair, tendon repair, bladder repair, and ophthalmic procedures.
• the placental compositions may also be applied or administered to treat wounds or injuries associated with other indications, including, but not limited to, osteoarthritis, inflammatory conditions (e.g., tennis elbow), bone defects, bone repair, and connective tissue repair.
• the wound site is associated with surgery of the spine and the stromal side of the placental composition is applied to the dura (e.g., the stromal side facing the dura).
• the stromal side of the placental composition is applied to the dura (e.g., the stromal side facing the dura).
• a placental composition of the present technology can optionally be used to reduce adhesion or fibrosis of a wound. Postoperative fibrosis is a natural consequence of all surgical wound healing.
• postoperative peridural adhesion results in tethering, traction, and compression of the thecal sac and nerve roots, which cause a recurrence of hyperesthesia that typically manifests a few months after laminectomy surgery.
• Repeated surgery for removal of scar tissue is associated with poor outcome and increased risk of injury because of the difficulty of identifying neural structures that are surrounded by scar tissue. Therefore, experimental and clinical studies have primarily focused on preventing the adhesion of scar tissue to the dura mater and nerve roots.
• Spinal adhesions have been implicated as a major contributing factor in failure of spine surgery. Fibrotic scar tissue can cause compression and tethering of nerve roots, which can be associated with recurrent pain and physical impairment.
• the placental compositions disclosed herein are useful in treating a number of wounds including: tendon repair, cartilage repair (e.g., femoral condyle, tibial plateau), ACL replacement at the tunnel/bone interface, PCL tendon repair, dental tissue augmentation, fistulas (e.g., Crohn's disease, G-tube, tracheoesophogeal), missing tissue at adhesion barriers (e.g., nasal septum repair, vaginal wall repair, abdominal wall repair, tumor resection), dermal wounds (e.g., partial thickness burns, toxic epidermal necrolysis, epidermolysis bullosa, pyoderma gangrenosum, ulcers e.g., diabetic ulcers (e.g., foot), venous leg ulcers), surgical wounds, periosteum replacement, keloids, organ lacerations, epithelial defects, and repair or replacement of a tympanic membrane.
• tendon repair
• compositions may be used to treat ocular wounds or injuries.
• Ocular wounds may be the result of inflammation, injury or surgery.
• the disclosure provides a method of treating an inflammatory ocular condition in a subject comprising administering to the subject a composition as disclosed herein, wherein the administration provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to treat the inflammatory ocular condition.
• the method can comprise administration of the membrane using any technique that may be directed to promote epithelialization, reduce pain, and/or to generally reduce inflammation of eye tissue.
• the method may be associated with eye surgery (e.g., photorefractive keratectomy (PRK)), eye trauma (e.g., lacerations, burns, or scrapes), or an eye disease that is characterized by inflammation or the treatment of which may result in an amount of inflammation in ocular tissue.
• eye surgery e.g., photorefractive keratectomy (PRK)
• eye trauma e.g., lacerations, burns, or scrapes
• an eye disease that is characterized by inflammation or the treatment of which may result in an amount of inflammation in ocular tissue.
• Non-limiting examples of indications that include an "inflammatory ocular condition" encompassed by the method include general repair/reconstruction of the corneal or conjunctiva surface(s) such as, for example, persistent epithelial defects; corneal ulceration; corneal transplant; descemetocele; corneal perforations; defects following excision of epithelial or subepithelial lesions or tumors (conjunctival tumors, conjunctival intraeptithelial neoplasia, subepithelial lesions, band keratopathy, scars, conjunctival folds parallel to the edges of eyelids); acute chemical burns; acute keratitis; painful bullous keratopathy; partial or complete limbal stem cell deficiency (with stem cell grafting); acute Stevens-Johnson syndrome; symbelpharon; fornix reconstruction; anophthalmia; bleb revisions; scleral thinning; and pterygium (see, e.g
• composition described may provide therapeutic amounts of tissue components (e.g., cells, therapeutic factors, extracellular matrix components and combinations thereof) that are effective to promote in vivo or in vitro:
• In vitro describes the experiments and/or procedures performed outside of the living organism (e.g., under tissue culture conditions using artificial culture medium), including, but not limited to, culture expansion of cells.
• In vivo describes experiments and/or procedures performed within an organism, for example, an animal or human.
• composition describe may also provide a therapeutic benefit, wherein the composition provides one or more placental cells, therapeutic factors, extracellular matrix components or combinations thereof in an amount effective to:
• the compositions are effective to stimulate tissue regeneration.
• Tissue regeneration may occur directly or indirectly by the application of the compositions described herein.
• Tissue regeneration may be directly regenerating the tissue damaged in a wound, injury or defect.
• tissue regeneration may also include indirect regeneration of tissue by stimulating the expression of therapeutic factors then stimulate the production of new tissue.
• Tissue regeneration may occur at, around or in the site of the wound or tissue injury.
• the compositions may also be used to promote angiogenesis in a subject in need thereof, for example, near, around, in or at the site of a wound or tissue injury.
• Compositions may promote the upregulation or secretion of angiogenic promoting growth factors, such as, for example, vascular endothelial growth factor (VEGF) and basic Fibroblast growth factor (bFGF).
• VEGF vascular endothelial growth factor
• bFGF basic Fibroblast growth factor
• compositions may be used to reduce inflammation. Reduction in inflammation may be shown by the increase in the expression of one or more antiinflammatory cytokines. Further, reduction in inflammation may be shown by the decrease in pro-inflammatory cytokines.
• compositions may also provide a reduction in the amount and/or activity of reactive oxygen species or an increase in the amount and/or activity of anti-oxidant agents.
• compositions may either directly or may indirectly promote one or more of: (i) a reduction of the amount and/or activity of pro-inflammatory cytokines; (ii) an increase in the amount and/or activity of anti-inflammatory cytokines; (iii) a reduction of the amount and/or activity of reactive oxygen species; (iv) an increase in the amount and/or activity of anti-oxidant agents; (v) a reduction of the amount and/or activity of proteases; (vi) an increase in cell proliferation; (vii) an increase in angiogenesis; and/or (viii) an increase in cell migration to the wound.
• the placental compositions disclosed herein can also be used with a carrier to form a composition, for example, a biocompatible scaffold.
• a carrier for example, a biocompatible scaffold.
• the placental compositions disclosed herein can be applied to a carrier. It would be appreciated by one skilled in the art that any suitable biocompatible scaffold, carrier or bone grafting material may be used.
• Suitable biocompatible scaffolds and/or suitable carriers include, but are not limited to, for example, allografts, autografts, xenografts, ceramics, bioglass, calcium sulphate, demineralized bone matrix, coral, collagen, graft composites, chondronic scaffolds, synthetic scaffolds of all types, natural/biological scaffolds of all types and the like (e.g., calcium phosphates, hydroxyapatite and tricalcium phosphate, collagen/ceramic composite, PCL, PLLA,PLGA, PEG, PGA, alginates, silk, collagen, dextran gelatin, elastin, agarose, chitosan, hyaluronan, HA-TCP-Collagen, GraftJacket®, Alloderm®, PriMatrix® and others). Types thereof include, but are not limited to, other configurations such as sponges, foams, films, sheets, gels.
• Suitable carriers include but are not limited to recombinant molecules such as Bone Morphogenic Protein (e.g., Infuse ((rhBMP-2), Infuse® Bone Graft and the like.
• Suitable autografts include but are not limited to bone (local bone or other), platelet rich plasma (PRP), bone marrow aspirate (BMA), adipose tissue and the like.
• the composition containing a carrier may be used for surgical repair of tissues, for example, bone repair (e.g., spinal fusions), tendon repair, cartilage repair and the like.
• kits for the treatment of a tissue wound or defect comprising at least one dose of the placental composition of the present technology in a pharmaceutically acceptable container.
• suitable containers include vials, pouches, bags, bottles, tubes and syringes.
• the kit can further comprise additives, such as, for example, antibiotics, emollients, humectants, anti-oxidants, preservatives, therapeutics, or combinations thereof.
• the kit can further include instructions for use of the kit.
• Other components of the kit can include, for example, a basin, bandages, dressings, adhesives, tools, scissors, scalpels, catheters, or combinations thereof.
• a whole placenta was obtained from a registered tissue bank after informed consent.
• the placenta was placed, with the maternal surface (rough surface) face down, on a sterile tray.
• the amniotic-chorionic membrane was cut and removed from the placenta.
• the chorionic membrane was then separated from the amnion and washed twice in D-PBS.
• the chorionic membrane was then soaked in an anticoagulant (ACD-A) solution to remove blood clots and then washed again in D-PBS.
• ACD-A anticoagulant
• the chorionic membrane was then digested by incubation with dispase for 30 min at 37 °C.
• the trophoblast layer was mechanically removed by scraping with fingers and the chorion was washed again in D-PBS.
• chorionic membrane was then weighed and incubated for 24 hours in an antibiotic cocktail, and washed again in D-PBS.
• Example 2 Obtaining a Placental Composition by Digestion and Homogenization
• a chorion membrane obtained from Example 1 was digested by incubation in 200 ml_ of a collagenase type II solution (300 U/mL in DMEM) for 10 min at 37 °C. The remaining un-digested chorionic membrane was then removed (the tissue fraction), leaving a digestion suspension containing collagenase and placental cells (the cellular fraction).
• the volume and container for digestion was determined based on the need to provide a suitable digestion environment for the tissue once placed on a shaker.
• the digestion was carried out on a standard plate shaker set at moderate speed in a 37 ⁇ € cell culture incubator.
• the cellular fraction comprising placental cells was centrifuged at 913 rcf for 5 min to separate the digestive enzyme (collagenase type II) from the placental cells.
• This centrifugation step may enhance cell viability by preventing over-digestion and ensure that the enzyme is washed away before homogenizing the tissue.
• This centrifugation step pellets the cells without damaging them, allowing the collagenase type II to be removed as supernatant.
• tissue fraction was washed twice in D-PBS to remove residual digestion enzyme and placed in a homogenization container with 1 ml_ cryoprotectant (5% DMSO in saline) per gram of chorionic membrane. This volume was determined to be appropriate for diluting the chorion membrane enough to produce a dispersion of ideal consistency while maintaining protein concentration at clinically significant levels.
• the temperature of the chorionic membrane was reduced by placing the container on ice for greater than 10 min. The chorionic membrane was then homogenized twice at high speed for 5 sec using a tissue homogenizer to obtain a chorionic homogenate.
• the homogenate (from the tissue fraction) was combined with the viable isolated placental cells (from the cellular fraction) and mixed thoroughly to provide a placental composition.
• the placental composition may be used (e.g., for therapy) fresh or may first be preserved (e.g., cryogenically) for a period of time.
• Example 3 Obtaining a Placental Composition by Digestion and Mincing
• a chorion membrane obtained from Example 1 after antibiotic treatment for 18-84 hr was digested by incubation in 200 ml_ of a collagenase type II solution (300 U/mL in DMEM) for 10 ⁇ 2 min at 37°C. The remaining un-digested chorionic membrane was then removed (the tissue fraction), leaving a digestion suspension containing collagenase and placental cells (the cellular fraction).
• the volume and container for digestion was determined based on the need to provide a suitable digestion environment for the tissue once placed on a shaker.
• the digestion was carried out on a standard plate shaker set at moderate speed in a 37 ⁇ € cell culture incubator.
• the cellular fraction comprising placental cells was combined with chilled D-PBS and centrifuged at 913 rcf for 5 min to separate the digestive enzyme (collagenase type II) from the placental cells.
• This centrifugation step may enhance cell viability by preventing over-digestion and ensure that the enzyme is washed away.
• This centrifugation step pellets the cells without damaging them, allowing the collagenase type II to be removed as supernatant.
• tissue fraction was washed twice in chilled D-PBS to remove residual digestion enzyme and transferred into a chilled glass dish.
• the chorion membrane was cut into pieces with tissue scissors then minced into small pieces with two scalpel blades by cross-slicing.
• the minced tissue fraction was pulled into a large syringe, an 18 gauge needle was attached to the syringe, and the minced tissue fraction was expulsed back into the dish.
• the minced tissue fraction was pulled back into the syringe, an 18 gauge needle was attached to the syringe, and the minced tissue fraction was expulsed into the tube with the cellular fraction.
• the minced tissue fraction and cellular fraction were mixed thoroughly to provide a placental composition.
• 5% DMSO was added dropwise to the placental composition with gentle swirling, then 5% HSA was added dropwise to the placental composition with gentle swirling.
• Example 4 Obtaining a Placental Composition by Mincing with a Herb Mincer
• a chorion membrane obtained from Example 1 after antibiotic treatment for 18-84 hr was transferred to a chilled glass dish, washed to remove residual antibiotic solution, and minced with a herb mincer for 6 min to achieve small, uniformly-sized pieces.
• CM chorion membrane
• Example 5 Obtaining a Placental Composition by Mincing with a Mezzaluna
• a chorion membrane obtained from Example 1 after antibiotic treatment for 18-84 hr was transferred to a chilled glass dish and minced with a mezzaluna for 6 min to achieve small, uniformly-sized pieces.
• the minced tissue was pulled into a large syringe, a 16 gauge needle was attached to the syringe, and the minced tissue fraction was expulsed back into the dish.
• the minced tissue fraction was pulled back into the syringe, a 18 gauge needle was attached to the syringe, and the minced tissue fraction was expulsed into a tube with ice packs.
• 5% DMSO was added dropwise to the placental composition with gentle swirling, then 5% HSA was added dropwise to the placental composition with gentle swirling.
• a 10 min collagenase digestion was able to produce high numbers of viable cells.
• 00256 Placentas (D136, D137) were obtained and processed according to the procedure detailed in Example 1 and Example 2, except with a collagenase type II concentration of 244 U/mL, as described above.
• a cell count of each cellular fraction by trypan blue staining and counting using a hemocytometer, was performed immediately following digestion to determine the number of viable cells per gram of each tissue. The data are presented in Figure 1 .
• 00257 Surprisingly, a substantial population of cells was isolated by digestion of less than 1 hr (e.g., 10 min). Digesting the tissue for only 10 min allowed the loosening and removal of cells from the tissue without completely breaking up the tissue.
• a fresh placental composition of the present technology comprises surprisingly high cell viability.
• a placental composition of the present technology subjected to a freeze-thaw cycle comprises surprisingly high cell viability.
• cell viability is retained surprisingly well after a freeze-thaw cycle.
• placental compositions of the present technology comprise a therapeutic profile of therapeutic factors.
• TSP1 TLTD TLTD TLTD Regulate TGF& activity, anti-angiogenic
• KGF (FGF-7 14.15 1 1 1 .58 45.72 Stimulate cell growth and migration
• EGF 0.42 3.72 1 .57 Stimulate cell growth and migration
• IGFBP1 5022.96 1227128.50 322596.69 Regulate IGF and its proliferative effects
• MMP1 Matrix Metalloproteinase 1
• MMP2 Matrix Metalloproteinase 1
• Tissue Inhibitors of MMPs (TIMP1 and TIMP2)
• Angiotensin-2 (Ang-2), Heparin-Bound Epidermal
• HB-EGF Growth Factor
• EGF EGF
• FGF-7 also known as EGF
• Keratinocyte Growth Factor-KGF Placenta
• PLGF growth and migration Growth Factor
• PEDF Thrombopoietin
• TGF-a Growth Factor-a
• PDGF Derived Growth Factors
• VEGF Vascular Endothelial Growth Factor
• HGF Hepatocyte Growth Factor
• G-CSF Granulocyte Colony-Stimulating Factor
• Interleukin 1 Receptor Antagonist IL-1 RA
• N-GAL Neutrophil Gelatinase-Associated Lipocalin
• LIF Leukemia Inhibitory Factor
• IGFBP1 Insulin-like Growth Factor Binding Protein
• Angiogenesis Angiotensin-2 (Ang-2), Fibroblast Growth
• bFGF Factor basic
• HB-EGF Epidermal Growth Factor
• EGF Keratinocyte Growth Factor
• KGF- also known as FGF-7 Platelet derived Growth Factors
• PDGF Platelet derived Growth Factors
• VEGF Vascular Endothelial Growth Factor
• HGF Hepatocyte Growth Factor
• PLGF Placental Growth Factor
• PEDF Pigment Epithelium Derived Factor
• TSP-2 Trombospondin-1
• EGF Epidermal Growth Factor
• KGF Keratinocyte Growth Factor
• Adiponectin (Acrp-30), Insulin Growth Factor 1 (IGF), Insulin-like growth factor binding protein (IGFBP 1 , 2, 3), Transforming Growth Factor a (TGFa), TGF- ⁇ , TGF-32
• Chemoattractant Stromal Cell Derived Factor 1 Beta (SDF- 1 b), bFGF, EGF, KGF
• MMP1 Matrix Metalloproteinase 1
• MMP2,3,7,8,9,10,13 Tissue Inhibitors of Function Therapeutic Factors and Protease Inhibitors MMPs (TIMP1 and 2), Alpha-2- macroglobulin, Fibronectin
• G- CSF Immunoregulatory Granulocyte Colony-Stimulating Factor
• IL- 1 RA Interlaukinl receptor antagonist
• LIF Leukemia Inhibitory Factor
• IFN-2a Interferon 2a
• PLAB Placental Bone Morphogenetic Protein
• FACS Fluorescence Activated Cell Sorting
• Example 1 Three placentas (D138, D139, and D140) were processed according to the procedure detailed in Example 1 , and each chorionic membrane was divided in half. Each piece of chorionic tissue was processed according to the procedure detailed in Example 2 with one of the following cryoprotectants:
• each fresh placental composition was counted for viable cells using a hemocytometer and trypan blue staining to distinguish live cells from dead cells. After freezing then thawing, each placental composition was counted for viable cells. The results are depicted in Figure 6.
• DMSO was a superior cryoprotectant compared to glycerol for both fresh and freeze/thawed placental composition.
• 5% DMSO represents a concentration that is suitable for preserving live cell numbers after freezing and thawing and is better for patient safety.
• Example 12 Cell Viability Three Days up to Three Months after Freezing
• Example 2 Three placentas were processed according to the procedure detailed in Example 1 , and each chorionic membrane was divided into four pieces of approximately equal weight ( ⁇ 1 g) prior to antibiotic treatment. One quarter was used to manufacture the composition following Example 2 (Control) and frozen in 5% DMSO in saline. The remaining three pieces were processed following Example 2, except that the tissue fraction was cut into small pieces with scissors, and then minced with scalpels into a flowable consistency. Saline was added, and the samples were frozen in 5% DMSO in saline, 5% DMSO and 1 % HSA in saline, or 10% DMSO and 5% HSA in saline.
• Group 1 Control group (tissue fraction homogenized), 5% DMSO in saline
• Group 2 Test group (tissue fraction minced), 5% DMSO in saline
• Group 3 Test group (tissue fraction minced), 5% DMSO and 1 % HSA in saline
• Group 4 Test group (tissue fraction minced), 10% DMSO and 5% HSA in saline
• Placental composition of each group was thawed 3 days, 1 month, 2 months, and 3 months after freezing and counted for viable and dead cells using a hemocytometer and trypan blue staining. Cell viability was calculated and results of the three placentas were averaged ( Figure 7).
• 00276 As depicted in Figure 7, mincing of the tissue fraction results in higher cell viability compared to homogenization of the tissue fraction. In addition, higher cell viability was observed for placental compositions which were frozen with a cryoprotectant solution containing HSA (Group 3 and 4 with 1 % to 5% HSA).
• chorionic tissues from three different donors were analyzed. The chorions were processed according to the procedure in Example 1 . Each chorionic membrane tissue was then washed twice to remove antibiotic solution and split into three pieces. Each piece of tissue was weighed to obtain an initial weight (0 min) before being digested for 10 min, 20 min, or 30 min in collagenase type II solution (300 U/mL).
• the remaining tissue was separated from the collagenase type II solution containing the isolated cells by filtering through a 100 ⁇ pore cell filter. The separated tissue was then weighed while the collagenase type II solution containing digested cells was centrifuged. The resulting cell pellet was re-suspended in D- PBS and counted using a hemocytometer with trypan blue exclusion.
• Figure 8 also shows the number of cells released by collagenase digestion. After 10 minutes of incubation, at least some of cells were released.
• Example 3 The limited digestion method of Example 3 was tested for applicability when the placental tissue is amniotic tissue. The following procedure was performed:
• a Place amniotic tissue in homogenization container with a volume of cryoprotectant (ml_) equal to the weight of the amniotic membrane (g). For example, if the amniotic membrane weighs 25 g, place it in the homogenization container with 25 ml_ of cryoprotectant.
• ml_ volume of cryoprotectant
• preserved endogenous placental protein e.g., matrix proteins
• a therapeutically effective composition e.g., a composition with a therapeutically effective composition.
• VEGFC 1 14.35 220.27 167.31
• Chorionic tissue was obtained from placental tissue of 9 subjects and the cellular fractions (e.g., collagenase-released) and homogenized tissue fractions were assessed for the number of live cells using a hemocytometer and trypan blue exclusion.
• cellular fractions e.g., collagenase-released
• homogenized tissue fractions were assessed for the number of live cells using a hemocytometer and trypan blue exclusion.
• the methods of the present invention unexpectedly preserve important therapeutic factors and live cells in the homogenized tissue fraction and also provide substantial numbers of live cells from the cellular fraction.
• Example 16 Angiogenic Growth Factors are Expressed for a Minimum of 14 days
• Placental compositions of the present technology demonstrate a durable effect desirable for wound healing treatments.
• the extracellular matrix and presence of viable cells within the placental composition derived from the chorionic membrane described in this technology allow for a cocktail of proteins that are known to be important for wound healing and angiogenesis to be present for at least 14 days.
• Placental compositions derived from the chorionic membrane and processed according to the procedure in Example 1 and Example 2 were thawed and plated onto tissue culture wells and incubated at 37 °C ⁇ 2°C for 3, 7, and 14 days. At each time point, a sample of the composition was collected and centrifuged at 16,000 rcf for 10 min to collect the supernatant. The supernatants were then tested by ELISA for bFGF and VEGF.
• Figure 10 illustrates the duration of two key wound healing proteins, bFGF and VEGF, at 3, 7 and 14 days. Although the expression of bFGF goes down with time, it should be noted that significant levels of bFGF was present even out to 14 days. Interestingly, the expression of VEGF increased with time, which could be due to continued active expression of VEGF from the viable cells within the placental composition derived from the chorionic membrane.
• the placental composition responded to the hypoxic environment by increasing the production of the angiogenic growth factor VEGF by 200%.
• VEGF and bFGF The content of the angiogenic growth factors VEGF and bFGF were measured before and after cryopreservation for three lots of placental composition (D144, D145, D146) processed according to the procedures in Example 1 and Example 2.
• a vial of each lot of the digested placental composition was reserved before cryopreservation at -80 °C and labeled as the fresh samples.
• the fresh samples were centrifuged at 16,000 rcf for 10 min in a microcentrifuge.
• the VEGF and bFGF concentrations were measured by ELISA.
• a vial of cryopreserved placental composition was thawed after at least 12 hours stored at -80 °C (cryopreserved samples).
• the cryopreserved samples were treated as described for the fresh samples above.
• the ELISA results are shown in Figure 12 and show that the content of both VEGF and bFGF was not altered by cryopreservation for all lots tested.
• IFN-2a Interferons
• TGF-33 Transforming Growth Factor-33
• IFN-2a Interferons
• TGF-33 Transforming Growth Factor-33
• IFN-2a is known to decrease collagen and fibronectin synthesis and fibroblast- mediated wound contracture.
• IFN-2a has been administered subcutaneously and shown to improve scar quality (Nedelec et al, Lab Clin Med 1995, 126:474).
• TGF- ⁇ 3 regulates the deposition of extracellular matrix and has been shown to decrease scar formation when injected in rodent cutaneous wound models.
• TGF- ⁇ 3 has been shown to improve scar appearance when injected at the wound site (Occleston et al., J Biomater Sci Polym Ed 2008, 19:1047).
• Placental compositions prepared as in Example 1 and Example 2 has been analyzed for the presence of IFN-2a and TGF-33. Briefly, placental composition derived from the chorionic membrane was thawed and centrifuged at 16,000 rcf to collect supernatants. Supernatants were analyzed on a commercially available ELISA kit from MabTech (IFN-2a) and R&D Systems (TGF-33). Figure 13 shows significant expression of IFN-2a and TGF-33 in placental compositions derived from the chorionic membrane.
• TGF-33 content of multiple lots of placental composition prepared as in Example 1 and Example 2 was measured.
• Four lots of cryopreserved placental composition were thawed and centrifuged at 16,000 rcf for 10 minutes in a microcentrifuge.
• the TGF-33 concentration was measured in supernatants by ELISA. Results are shown in Figure 14 shows expression of TGF- ⁇ 3 in placental compositions.
• Example 21 Tissue Reparative Proteins in Chorionic Placental Composition
• Placental compositions derived from the chorionic membrane and processed as in Example 1 and Example 2 were analyzed for the presence of proteins that are important in tissue repair (e.g., therapeutic factors or tissue repair proteins).
• Placental compositions derived from chorionic membranes described in this invention were analyzed for the presence of these tissue reparative proteins. Briefly, placental compositions derived from the chorionic membrane was incubated at 37 °C ⁇ 2 °C for 72 hrs. The compositions were centrifuged, and the supernatants were analyzed on commercially available ELISA kits from R&D Systems.
• FIG. 16 shows significant expression of BMP-2, BMP-4, BMP-7, PLAB, PLGF, and IGF-1 in several donors of placental compositions derived from chorionic membranes.
• BMP-2 and BMP-4 may stimulate differentiation of MSCs to osteoblasts in addition to promote cell growth ; placental BMP or PLAB is a novel member of the BMP family that is suggested to mediate embryonic development.
• IGF-1 insulin-like growth factor 1
• PLGF Placental derived growth factor
• Placental cells in optional embodiments of the present invention, are adherent, express specific cellular markers such as CD105 and lack expression of other markers such as CD45, and demonstrate the ability to differentiate into adipocytes, osteoblasts, and chondrocytes.
• Figure 16-A shows a representative image of passage 2 bone marrow MSCs, demonstrating the ability of the cells to adhere to tissue culture plastic.
• Figure 16-B a representative image of cells isolated and expanded from human chorion membrane is shown in Figure 16-B.
• 00308 Osteogenic differentiation capacity was demonstrated by staining the cultured cells with alkaline phosphatase labeling following the manufacturer's recommendations (BCIP/NBT Alkaline Phosphatase Substrate Kit IV, Vector Laboratories Cat. No. SK-5400).
• Alkaline phosphatase is an enzyme involved in bone mineralization (Allori et al., Tissue Engineering: Part B, 2008, 8:275), and its expression within cells is indicative of osteo- precursor cells (Majors et al., J Orthopaedic Res, 1997, 15:546). Staining for alkaline phosphatase is carried out through an enzymatic reaction with Bromo-4-Chloro-3'- Indolylphosphate p-Toluidine Salt (BCIP) and Nitro-Blue Tetrazolium Chloride (NBT). BCIP is hydrolyzed by alkaline phosphatase to form an intermediate that undergoes dimerization to produce an indigo dye. The NBT is reduced to the NBT-formazan by the two reducing equivalents generated by the dimerization. Together these reactions produce an intense, insoluble black-purple precipitate when reacted with alkaline phosphatase.
• BCIP Bromo-4-Chloro-3'- Indolylphosphate
• Figure 16-C shows a representative image of passage 2 cells isolated and expanded from placental composition derived from the chorionic membrane staining positively for alkaline phosphatase after osteoinduction.
• Placental composition produced as in Example 4 contains the placental cells within the small tissue pieces generated by mincing. In order to quantify these placental cells using a hemocytometer and trypan blue exclusion, the cells must first be released from the tissue pieces by collagenase digestion.
• Placental composition was tested for response to inflammation to mimic the inflammatory conditions found in chronic wounds.
• the inhibition of TNF-a production by PBMCs peripheral blood mononuclear cells
• PBMCs peripheral blood mononuclear cells
• Example 1 Two placentas processed as in Example 1 were each split in half before antibiotic treatment. One half of each was processed into placental composition as in Example 3 (digested placental composition) and half was processed into placental composition as in Example 4 (minced placental composition). The minced and digested placental compositions with their different configurations were compared for their respective ability to modulate inflammation.
• CD3 and CD28 were added to PBMCs to stimulate the production of inflammatory cytokines such as TNF-a.
• the PBMCs were then cultured with either minced or digested placental composition.
• stimulated PBMCs were cultured in DMEM.
• PBMCs without stimulatory CD3 and CD28 were cultured in DMEM.
• the cultures were incubated for 60-84 hr until the positive control culture displayed PBMC aggregation.
• the culture samples were collected and centrifuged at 16,000 rcf for 10 min.
• the supernatants were collected and tested for TNF- a content by ELISA (results shown in Figure 18).
• Minced placental product processed by Example 1 and Example 4, was tested in an elastase inhibition assay for its ability to mediate protease activity.
• Enzymatic substrate hydrolysis results in an increase in absorbance. Enzymatic substrate hydrolysis was lowered in minced placental composition as compared to the positive control confirming the ability of minced placental composition to regulate the chronic wound environment through inhibition of proteases.
• Example 3 Two placentas processed as in Example 1 were each split in half before antibiotic treatment. One half of each was processed into placental composition as in Example 3 (digested placental composition) and half was processed into placental composition as in Example 4 (minced placental composition).
• minced and digested placental compositions contain VEGF, though minced composition contains a greater quantity for both tested lots. Because minced placental composition is not partially digested in collagenase as placental composition prepared as in Example 2 or Example 3, minced placental composition likely retains a greater quantity of native growth factors and extracellular matrix proteins.
• placental compositions were subjected to lysis in guanidine HCI.
• placental compositions were subjected to lysis in guanidine HCI.
• Two placentas processed as in Example 1 were each split in half before antibiotic treatment. One half of each was processed into placental composition as in Example 3 (digested placental composition) and half was processed into placental composition as in Example 4 (minced placental composition).
• a protease inhibitor tablet (Complete protease inhibitor cocktail tablets, Roche # 04693124001 ) was added to 10 mL of chilled 8 M Guanidine HCI (hereafter referred to as GuHCI). Minced and digested placental compositions were thawed and centrifuged at 16,000 rcf for 12 min. Supernatants were removed and moved into fresh tubes and placed on ice. The tubes containing the cell/tissue pellets were snap frozen by placing in liquid nitrogen for 5 min. A chilled lysis bead and GuHCI to a final concentration of 4M was added to each pellet. The pellets were placed in the chilled chamber of the Tissue Lyser, and the samples were lysed for 6 min at 50 Hz.
• GuHCI Guanidine HCI
• placental compositions were subjected to lysis in tissue extraction buffer. Previous experiments showed the bFGF ELISA to be incompatible with guanidine HCI treatment so alternative lysis buffers were explored.
• compositions were cultured for two weeks and VEGF content was measured.
• Example 3 Two placentas processed as in Example 1 were each split in half before antibiotic treatment. One half of each was processed into placental composition as in Example 3 (digested placental composition) and half was processed into placental composition as in Example 4 (minced placental composition).
• 00335 The cell proliferation capabilities of minced placental compositions were tested. Placental cells were seeded and cultured for 14 days. 00336 Vials of placental composition, prepared as in Example 1 and Example 4, were thawed and digested for 20 min with rocking at 37 °C. Each sample was digested either in 250 U/mL Serva type II collagenase or 250 U/mL Worthington type I I collagenase. The digested compositions were poured over 100 ⁇ cell strainers, and the strainers were washed with DMEM. The digested compositions were centrifuged for 10 ⁇ min at 41 1 rcf. The supernatants were removed and re-suspended in DMEM. Each cell suspension was counted with a hematocytometer by trypan blue exclusion.
• Carrier materials were placed in individual wells of a 24-well Nunclon multidish.
• the carrier materials included HA-TCP-Collagen Foam (1 cm x 1 cm piece) and TranZgraft cancellous granules.
• Minced placental composition produced as in Example 1 and Example 4, was thawed and added to each of to the different materials.
• D-PBS was added to the samples and the samples were left at room temperature for one hour to mimic the maximum amount of time surgeons are recommended to leave placental composition mixed with carrier after thawing. The liquid was removed from the wells.
• the materials were stained for viable and non-viable cells using the LIVE/DEAD® assay (Life Technologies, Grand Island, NY).
• Tissue piece size of a placental composition prepared as in Example 1 and Example 4 (minced with herb mincer) was compared to a placental composition prepared as in Example 1 and Example 5 (minced with mezzaluna).
• Final composition was thawed, and 10 ⁇ were transferred to a hematocytometer. 5 ⁇ trypan blue was added, and a circular coverglass was placed on top of the cell suspension.
• the size of tissue pieces for final composition prepared using a herb mincer or mezzaluna were compared.
• Tissue pieces of chorion minced with the mezzaluna were significantly larger than tissue pieces of chorion minced by the herb mincer (see Figure 26).
• 00348 Diagnosis. L5-S1 herniation with right foraminal narrowing and collapse. Right L5 and S1 radiculopathy with mechanical instability of L5-S1 . Right ankle weakness concordant to disc herniation and right lower extremity radiculomyelopathy. 00349 Surgical Procedure. Approximately 12 months post-injury, the right sided interpedicular space at L5-S1 was microsurgically dissected and the disc material was removed via facetectomy, foraminotomy and microdiscetomy. Preparation of the bone graft was completed which consisted of 15 cc of demineralized bone matrix (DBM) mixed with 1 ml_ of placental composition.
• DBM demineralized bone matrix
• Both grafting materials are radiolucent at implantation.
• the endplates were decorticated and an oblique 8 mm T- PAL PEEK spacer was packed with 5 cc of DBM+placental composition and placed transforaminally. Extensive decortication along the left dorsolateral mass of L5-S1 was completed through a microsurgical dissection. The remaining 10 cc of DBM+placental composition was applied to the left dorsolateral mass for onlay fusion. Rod and cap assembly was completed.
• Coronal and sagittal CTs at 6 months post-surgery show evidence of early fusion through the left facet joint and early interbody trabecular bone formation.
• the interbody bone graft and hardware are in optimal position.
• CT thin sections at an early time point can identify non-union but is not as sensitive at identifying fusion.
• Early bone fusion can typically be visualized on CT in as little as 6-12 months minimally (Williams A, Gornet M, Burkus K. CT Evaluation of Lumbar Interbody Fusion: Current Concepts. AJNR Am J Neuroradiol 2005; 26:2057-2066).
• Example 35 Compositions for Use in Treating Tunnel Wounds
• a patient with a tunnel wound is treated by administration once a week with 1 mL of the cryopreserved placental composition for at least 4 to 6 weeks. Treatment is stopped when there is re-epithelialization and closure of the wound.
• fetal macrophages present in the amnion and chorion are a major source of tissue immunogenicity.
• tissue immunogenicity Without being bound by theory, the present inventors believe that removal of CD14+ fetal macrophages from placental membrane and compositions prevents activation of lymphocytes and decreases the level of inflammatory cytokine secretion and tissue immunogenicity.
• Macrophages in fetal placental membranes respond to bacterial LPS by secretion of inflammatory cytokines such as TNF-a. Therefore, secretion of TNF-a in response to LPS is used here to characterize tissue immunogenicity of placental membranes at each critical manufacturing step.
• T trophoblast
• ACT amnion with chorio trophoblast
• CT choriotrophoblast
• CM chorion
• AM amnion
• Tissue culture supernatants were then collected and tested for the presence of TNF-a using a TNF-a ELISA kit (R&D Systems) according to the manufacturer's protocol.
• hPBMCs Human hPBMCs (SeraCare) known to contain monocytes responding to LPS by secretion of high levels of TNF-a were used as a positive control. hPBMCs and placental tissues without LPS were also included as controls in the analysis. In this assay, TNF-a detected in the culture medium from greater than 70 pg/cm 2 (corresponding to 280 pg/mL) for both spontaneous and LPS-induced TNF-a secretion was considered immunogenic (Fortunato, et al.1996).
• the manufacturing process serially reduces immunogenicity of the placental product.
• AM and CM had only 23.5 and 40pg/ml TNF-a secretion as compared to ACT and CT at 1397.1 and 917.2 pg/ml, respectively.
• Tissues cultured in medium without LPS show the basal level of TNF-a secretion.
• PBMCs which are known to secrete high levels of TNF, were used as a positive control.
• CT Choriotrophoblast membranes
• Figure 28 CT cells were cultured with PBMCs for 4 days.
• IL-2Ra was measured in cell lysates as a marker of T-cell activation.
• Positive control a mixture of PBMCs derived from 2 different donors. Results of this assay, as seen in Figure 28, showed a correlation with the MLR data: tissues that produce high levels of TNF-a in response to LPS are immunogenic in the MLR assay
• the low levels of TNF-a and the absence of the response to LPS by AM and CM indicates the exemplary cryopreservation method described in the current technology eliminates viable functional macrophages from the amniotic and chorionic membranes, which ensures the safety of such an allogeneic product.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• General Health & Medical Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Engineering & Computer Science (AREA)
• Cell Biology (AREA)
• Epidemiology (AREA)
• Developmental Biology & Embryology (AREA)
• Immunology (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• Virology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Organic Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Pregnancy & Childbirth (AREA)
• Dermatology (AREA)
• Reproductive Health (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Rheumatology (AREA)
• Gastroenterology & Hepatology (AREA)
• Surgery (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Medicinal Preparation (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (7)

Applications Claiming Priority (1)

Publications (1)

Family

ID=54392804

Family Applications (1)

Country Status (7)

Cited By (13)

Families Citing this family (1)

Citations (3)

Family Cites Families (4)

• 2014
  • 2014-05-07 WO PCT/US2014/037201 patent/WO2015171142A1/en active Application Filing
  • 2014-05-07 EP EP14891336.1A patent/EP3139935A4/en not_active Withdrawn
  • 2014-05-07 KR KR1020167034174A patent/KR20160147058A/ko not_active Application Discontinuation
  • 2014-05-07 AU AU2014393402A patent/AU2014393402A1/en not_active Abandoned
  • 2014-05-07 JP JP2016566997A patent/JP2017514876A/ja active Pending
  • 2014-05-07 CA CA2948126A patent/CA2948126A1/en not_active Abandoned
  • 2014-05-07 SG SG11201609254YA patent/SG11201609254YA/en unknown

Patent Citations (4)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events